Fabrication of Microfluidic Devices for Continuously Monitoring Yeast Aging

For several decades, aging in Saccharomyces cerevisiae has been studied in hopes of understanding its causes and identifying conserved pathways that also drive aging in multicellular eukaryotes. While the short lifespan and unicellular nature of budding yeast has allowed its aging process to be observed by dissecting mother cells away from daughter cells under a microscope, this technique does not allow continuous, high-resolution, and high-throughput studies to be performed. Here, we present a protocol for constructing microfluidic devices for studying yeast aging that are free from these limitations. Our approach uses multilayer photolithography and soft lithography with polydimethylsiloxane (PDMS) to construct microfluidic devices with distinct single-cell trapping regions as well as channels for supplying media and removing recently born daughter cells. By doing so, aging yeast cells can be imaged at scale for the entirety of their lifespans, and the dynamics of molecular processes within single cells can be simultaneously tracked using fluorescence microscopy. Key features This protocol requires access to a photolithography lab in a cleanroom facility. Photolithography process for patterning photoresist on silicon wafers with multiple different feature heights. Soft lithography process for making PDMS microfluidic devices from silicon wafer templates.

For several decades, aging in Saccharomyces cerevisiae has been studied in hopes of understanding its causes and identifying conserved pathways that also drive aging in multicellular eukaryotes. While the short lifespan and unicellular nature of budding yeast has allowed its aging process to be observed by dissecting mother cells away from daughter cells under a microscope, this technique does not allow continuous, high-resolution, and highthroughput studies to be performed. Here, we present a protocol for constructing microfluidic devices for studying yeast aging that are free from these limitations. Our approach uses multilayer photolithography and soft lithography with polydimethylsiloxane (PDMS) to construct microfluidic devices with distinct single-cell trapping regions as well as channels for supplying media and removing recently born daughter cells. By doing so, aging yeast cells can be imaged at scale for the entirety of their lifespans, and the dynamics of molecular processes within single cells can be simultaneously tracked using fluorescence microscopy.

Background
Progress in aging research has been greatly accelerated in recent years due to the development of new tools and techniques for single-cell analysis (Dulken et al., 2019;Li et al., 2020;Tabula Muris, 2020;Trapp et al., 2021;Roux et al., 2022). Microfluidic technologies play pivotal roles in these applications, as they allow single cells to be captured in droplets for sequencing (Matuła et al., 2020) or isolated for long-term imaging (Allard et al., 2022). For studying replicative aging in Saccharomyces cerevisiae, the traditional method of manual microdissection of mother and daughter cells in order to count replicative lifespan has been largely supplanted by the use of microfluidic devices, which efficiently trap individual mother cells in place while removing newly budded daughter cells Recently, our group has used these devices to reveal that cells undergoing one of the aging trajectories display a reduction in protein homeostasis, with RNA binding proteins aggregating after diminished control of chromatin silencing at the rDNA locus . Here, we report a detailed protocol for constructing the microfluidic devices for yeast replicative aging experiments that have facilitated the discovery and characterization of these trajectories.

Materials and reagents
Software 1. AutoCAD from AutoDesk (https://www.autodesk.com/products/autocad). Requires license but this is free to download for students and educators.

Procedure
A. Obtain chrome glass masks for photolithography 1. As part of this protocol, we have created an AutoCAD file with only the designs for the microfluidic devices used in Paxman et al. (2022) and included it in the Supplement. See Figure 1 for the design of the microfluidic device. Email the .dwg AutoCAD file to a company such as HTA photomask, which makes chrome glass masks. Order two 5 × 5 inch chrome photomasks made of quartz glass. Use ± 0.25 μm tolerance for the first mask containing the cell traps and ± 0.5 μm tolerance for the second mask containing the media channels. Have the closed objects on the .dwg file printed as clear on the mask and have the printing as right reading down with the chrome side facing down. c. Rinse wafer with isopropanol and then sonicate in isopropanol for 5 min. d. Rinse wafer with deionized water. Note: While this step may be useful for certain kinds of silicon wafers, we have found it to be unnecessary if using the wafers included in the Materials and Reagents section (100 mm wafers). Therefore, if using these wafers, we recommend skipping this step.

Dehydration bake:
a. Bake the wafer at 150 °C for 15 min followed by a 15 min cool down to room temperature (RT).

C. Photolithography for cell trapping layer (Layer 1)
1. See Figure 2 for an overview of the process for Layer 1.  photoresists recommend a first step at a speed of 500 rpm and acceleration of 100 rpm/s. However, since the target height of the cell traps is 4.5 μm, we found it more reliable to achieve this height by omitting this first step and spinning immediately at the high speeds than by following the recommended protocol from the manufacturer, in which we found it difficult to reliably spin this photoresist down to less than 5 μm. Therefore, in our experience, modulating the initial acceleration is a powerful method for tuning the photoresist height. 5. When spin coating has been completed, soft bake the wafer at 65 °C for 9 min. Place the lid of a Pyrex dish over the wafer while it is on the hot plate. Position the lid so that it slightly hangs over the end of the hotplate. When time is up, remove the Pyrex dish lid and use wafer tweezers to remove the wafer from the hot plate. Place the wafer on the lid and allow it to cool down to RT for 3 min. Note: Placing the lid over the wafer is done to give the wafer a surface of equal temperature to cool on, so that it does not cool too fast. This is done for all baking steps in this protocol. 6. Expose the wafer to UV light on the EVG620 using the Layer 1 chrome glass mask without a filter in place at 75 mJ/cm 2 using the vacuum contact setting. 7. Remove the wafer from the mask aligner. 8. Perform a 2 min post-exposure bake at 95 °C. Arrange the Pyrex dish lid over the wafer as before. Allow 3 min to cool down to RT. 9. Pour SU-8 Developer in a large Pyrex dish and develop the wafer for 2 min. During this time, gently shake the dish back and forth and side to side. After 2 min, take the wafer out of the dish and rinse it with fresh developer for ~10 s; then, rinse with isopropanol for ~20 s. Blow the wafer dry using a nitrogen spray gun. 10. Hard bake the wafer at 95 °C for 5 min with a 3 min cool down to RT afterward. 11. Measure the height of the layer using a profilometer and assess feature integrity under a microscope. While there is inherent variability in the spin coating process, it is important to verify that the measured height is close to that of the design. Note: When assessing the first layer height and feature integrity under an upright microscope, ensure that trap dimensions are close to those shown in Figure 1C and 1D and that there are no cracks or dents in the photoresist layer. Successfully built wafers should have dimensions within ±10% of the design specifications for the first layer.

D. Photolithography for media channel layer (Layer 2)
1. Tape over the alignment markers on the wafer (Figure 3). To do this, cut out a piece of Kapton tape and cut out a small square from the adhesive labels that is large enough the cover the alignment markers. Using tweezers, place the sticky side of the cut-out label square onto the sticky side of the Kapton tape. Place this over the alignment markers on the wafer so that the non-sticky side of the label is covering the alignment markers. Use a tweezer to press down on the tape surrounding the alignment markers so that it sticks to the wafer. Trim Kapton tape hanging off the wafer but leave enough excess to grab with a tweezer (tape will be removed after spin coating). 3. Set the time on the spin coater to 40 s and set the spin speed to 1,450 rpm. For the acceleration, select the preset value that is closest to 1,450 rpm/s, so that the wafer will reach its desired spin speed in 1 s. Note: In the original build for this wafer, these settings were used to maintain consistency with the method of spinning the first layer. However, in later builds we have switched to a more standard approach for achieving a 15 μm layer by using SU-8 2015 and spinning in two steps: step 1 is at 500 rpm with a 136 rpm/s acceleration for 10 s, and step 2 at 3,000 rpm with a 272 rpm/s acceleration for 40 s. We recommend this latter approach for the build. 4. With a tweezer, carefully remove the tape over the alignment markers on the wafer. 5. Soft bake the wafer at 65 °C for 15 min with a 3 min cool down to RT afterward. 6. Alignment of Layer 1 to the mask for Layer 2: a. See Figure 4 for the design of the alignment markers, the goal of the alignment process, and example images during alignment. Align Layer 1 and Layer 2 alignment markers by adjusting the X, Y, and Theta knobs on the EVG620 mask aligner. The most efficient way to accomplish this is to first tune the focus and position of the lenses on the EVG620 to locate the alignment markers on the Layer 2 mask. Then, turn the knobs to locate the alignment markers on the wafer. Then, to align the two sets of markers, correct half in the Y direction and half in the Theta direction until all sets of squares are fully aligned (see Figure 4). Perform any intermittent corrections in the X direction as needed during this process.  Progressing views of alignment process. Markers on the wafer and the mask begin out of alignment (top) but are gradually put into correct alignment by tuning the X, Y, and Theta knobs on the mask aligner. The largest markers are aligned first (middle), followed by further improvement of alignment by correctly orienting the smallest set of markers (bottom), thereby completing the alignment process.

7.
Expose the wafer on the EVG620 using the Layer 2 chrome glass mask without a filter at 125 mJ/cm 2 using the hard contact setting. 8. Post-exposure bake at 95 °C for 5 min with a 3 min cool down to RT.

9
Published: Aug 05, 2023 9. Pour fresh SU-8 Developer into the large Pyrex dish and develop the wafer for approximately 5 min with gentle shaking. Rinse with fresh developer and then isopropanol and blow dry with a nitrogen spray gun. 10. Hard bake at 95 °C for 5 min with a 3 min cool down to RT. 11. Measure the height of the media channels with a profilometer and assess feature integrity of the final wafer using an upright microscope ( Figure 5A and 5B). Note: Ensure that the photoresist layer is free of large cracks and dents when analyzing the wafer under an upright microscope. Error tolerability in height for the second layer is larger than that for the first; however, aim for ±20% from the design specifications. Importantly, make sure the traps are well aligned between the channels as in Figure 5B.